Project Summary Advanced estrogen receptor (ER)-positive breast cancers are initially responsive to multiple therapeutic interventions, but they ultimately develop drug resistance and disseminate to multiple metastatic sites. Circulating tumor cells (CTCs) underlie the blood-borne spread of cancer and they also provide a noninvasive source to sample, monitor and analyze tumor evolution in real time, as patients develop progressively resistant disease with new metastatic lesions. To enable the detailed molecular study of CTCs, which are extremely rare cells in the circulation, we have made use of microfluidic platforms that efficiently deplete normal hematopoietic cells from blood specimens, leaving behind an enriched population of intact CTCs, some of which remain viable. During the past funding period, we established a panel of patient-derived breast cancer CTC cell lines (Yu et al., Science 2014), which provide a window into critical and poorly understood properties of advanced breast cancer, with significant clinical implications. We demonstrated that these heterogeneous ER+ drug-resistant breast cancer cells contain distinct phenotypes, with a HER2-expressing proliferative state interconverting spontaneously with a Notch1- driven drug resistant state (Jordan et al., Nature 2016). In Aim 1, we will build on this observation to define the likely epigenetic mechanisms that modulate this phenotype conversion. Using live-reporter constructs, we will isolate single cells as they switch between phenotypes to define early transcriptional changes, and in bar-coded pooled knockdown screens, we will test how chromatin modulators affect this phenotype switch, both spontaneously and following the dramatically enhanced reactive oxygen species (ROS)- mediated conversion that we have observed. In Aim 2, we will study another unexpected observation made with cultured breast CTCs, namely their acquired quiescence following direct intravascular inoculation and dissemination to the lung. While a 200 CTC inoculum can initiate tumorigenesis in the mammary gland, tail vein inoculation of 200,000 CTCs leads to non-proliferative single cells throughout the lung, an observation that may be linked to ROS stress experienced by these cells in the bloodstream (Zheng et al., Nature Comm, 2017). We have used pooled bar-coded knockdown construct libraries of chromatin modulators to uncover candidate regulators that are enriched as CTCs eventually initiate proliferation in the lung, and these will be tested individually and in combination, validated in multiple CTC lines, and matched with RNA seq transcriptomes of early stages in the transition from quiescence to early proliferative metastatic lesions. In Aim III, we will examine organ-specific pathways that enable proliferation of breast CTCs in the brain versus bone or liver. By serial inoculation, we have generated derivative lines of CTCs that grow efficiently following direct implantation in brain, compared with parental cells which exhibit a prolonged delay. Using both RNA sequencing and whole proteome mass spectrometry, we will identify modulators of organ-predominant metastasis, which will then be validated through functional assays and correlated with primary CTCs from patients with organ-predominant metastases. All together, these experiments will use patient-derived cultures of metastatic precursors to better understand and ultimately target breast cancer progression.